The present invention relates to a process for amplifying a sequence of a target nucleic acid.
The document EP-A-0 285 057 discloses a process for treating a nucleotide, consisting in introducing, into one of the constituent elements of said nucleotide, namely the sugar, the purine or pyrimidine base and the phosphate groups, a functional group having various uses, in particular those of labeling. The resulting nucleotide, which has thus been treated, can, according to this document, be incorporated into a polynucleotide, in particular in double-stranded form, without the double helix being destabilized by the presence of this nucleotide.
However, in this case, the functional groups which are attached to the nucleotide in accordance with this prior art exhibit a variety of phenomena, such as steric hindrance, hydrophobic interactions or complexing phenomena, which prevent the polynucleotide which has incorporated said treated nucleotide from being recognized by the majority of the enzymes which would recognize the corresponding polynucleotide into which said treated nucleotide had not been incorporated.
The document WO-A-92/00989 proposes a process which uses a protein for labeling a nucleic acid sequence, in particular for obtaining a labeled probe, by amplifying a target nucleic acid. According to this process, use is made of modified nucleotides which differ from the natural nucleotides by the presence of a reactive function, amplification is carried out in order to obtain a prefunctionalized probe, and the resulting probe is reacted, by way of the reactive functions, with a protein label. To this end, the reactive function of the modified nucleotide is a nucleophilic function which is selected from thiol functions, which may, where appropriate, be protected, and amino functions.
The drawback of this process lies in the selected label which, because of its size, with the molecular weight being of the order of several thousand, will substantially limit the rate and/or yield of the covalent coupling between said label and said function, with this problem increasing with the number of modified nucleotides which are incorporated into the probe. What is more, the label can lead to a deceleration of the reactions in which the resulting probe is involved, in particular in a hybridization.
The present invention provides an amplification process which overcomes the previously mentioned drawbacks and which, in particular, does not disrupt the incorporation of the nucleotides and, as a consequence, does not have a significant influence on the yield and/or the sensitivity in a target amplification reaction, and which, furthermore, makes it possible to obtain excellent labeling of the amplification products by avoiding label instability phenomena which were previously associated with incorporation of the label.
Thus, the present invention relates to a process for amplifying a target nucleic acid sequence, according to which:
at least the following are available: the sequence of a target nucleic acid, at least one oligonucleotide primer which is specific for the target sequence, and one or more nucleotide enzyme activities,
the target sequence is amplified under nucleotides is a prefunctonalized nucleotide which differs from the other nucleotides at least by the presence of at least one covalently reactive function, which is unprotected and which is arranged in at least one predetermined site on the base of said nucleotide, in order to obtain a prefunctionalized amplification product which includes at least one said prefunctionalized nucleotide.
According to an advantageous process of the invention, this process additionally comprises the following steps:
a reagent is available which comprises a covalently anti-reactive function, which is specific for the reactive function of the prefunctionalized nucleotide, and a functional group, and
the prefunctionalized amplification product is reacted, directly or indirectly, with the reagent in order to obtain a functionalized amplification product.
Some of the terms employed in the present description are defined below, after which the invention is explained in detail.
Nucleotide according to the invention is understood as being a natural or modified nucleotide monomer as defined below.
Thus, the nucleotide monomer can be a natural nucleic acid nucleotide whose constituent elements are a sugar, a phosphate group and a nitrogen base; the sugar is ribose in RNA and is 2xe2x80x2-deoxyribose in DNA; depending on whether the nucleic acid is DNA or RNA, the nitrogen base is selected from adenine, guanine, uracil, cytosine and thymine; or a nucleotide which is modified in at least one of the three constituent elements; by way of example, the modification can take place at the level of the bases, generating modified products such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine or bromo-5-deoxyuridine, and any other modified base which permits hybridization, at the level of the sugar, namely, for example, replacement of at least one deoxyribose by an analog (for example: P. E. Nielsen et al., Science, 254, 1497-1500 (1991)), at the level of the phosphate group, for example boronate, alkylphosphonate or phosphorothioate derivatives.
A protective group is understood as being the groups which are conventionally employed in the chemical synthesis of nucleosides, nucleotides and oligonucleotides (see, for example: Chemistry of Nucleosides and Nucleotides, edited by Leroy B. Townsend, Plenum Press, New York and London and Protocols for Oligonucleotides and Analogs, Synthesis and Properties, edited by Sudhir Agrawal, Humana Press, Totowa, N.J.).
A labeling functional group of the invention is a molecule which is capable of directly or indirectly generating a detectable signal. The group is, in particular, selected from radioactive isotopes, enzymes which are selected, in particular, from peroxidase, alkaline phosphatase and b-galactosidase, and those enzymes which are capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate, chromophoric, chromogenic, fluorophoric, fluorigenic or luminescent chemical compounds, nucleotide base analogs and ligands such as biotin.
When the label is unable to generate a signal directly, for example when the label is an enzyme, it is necessary to add a visualizing substance, for example a substrate which corresponds to the enzyme, with the enzyme/substrate reaction generating a detectable complex for example a chromogenic or luminescent compound. By way of example, the visualizing reagent can be ortho-phenylenediamine or 4-methylumbelliferyl phosphate.
The covalently reactive function of the prefunctionalized nucleotide and the anti-reactive function of the reagent are electrophilic and nucleophilic organic chemical functions, respectively, or vice versa.
The electrophilic organic chemical function is advantageously selected from the aldehyde, activated ester, carboxylic acid, isothiocyanate, haloacyl derivatives and sulfonyl chloride functions.
The nucleophilic organic chemical function is advantageously selected from the amino, thiol, oxyamino, hydrazine and hydrazide functions; it is preferably the alkoxyamino function.
According to one variant of the invention, the covalently reactive function of the modified nucleotide is attached to the base by way of a coupling arm and/or the covalently anti-reactive function of the reagent is attached to the functional group by way of a coupling arm.
The coupling arm is selected, in particular, from saturated or unsaturated hydrocarbon chains, which are interrupted, where appropriate, by amino, amido and oxy functions.
According to a preferred process, the covalently reactive function is the oxyamino function and the covalently anti-reactive function of the reagent is the aldehyde function, and this latter is linked to a labeling functional group such as a fluorescent or luminescent group.
Advantageously, the aldehyde function is linked to the functional group by the coupling arm xe2x80x94NHxe2x80x94CSxe2x80x94NHxe2x80x94(CH2)3xe2x80x94, and the functional group of the reagent is fluorescein.
The prefunctionalized nucleotide product can comprise one or more, identical or different, covalently reactive functions which are introduced by one or more nucleotides. Said covalently reactive functions can react with one or more, identical or different, reagents simultaneously or sequentially. The labeling functional groups of the functionalized product can be detected simultaneously or sequentially.
It will be understood that this labeling process can be applied to one or more prefunctionalized nucleotide products, in particular in order to differentiate prefunctionalized products which are derived from different targets.
The labeled functionalized amplification product can be detected qualitatively and/or quantitatively, in homogeneous or heterogeneous phase.
In homogeneous phase, the reagent and the prefunctionalized nucleotide product interact in the same liquid medium. In heterogeneous phase, the prefunctionalized product can be treated with the reagent before or after capture on a solid support, that is to say directly or indirectly. The capture on the solid support can be effected using known means, such as adsorption, covalency, in particular using covalently anti-reactive functions which are available on the surface of the solid support, or by means of hybridization using a polynucleotide compound.
The solid support, in all suitable forms, such as tube, cone, well, microtitration plate, sheet, chip or soluble polymer, is selected from polystyrenes, styrene-butadiene copolymers, styrene-butadiene copolymers mixed with polystyrenes, polypropylenes, polycarbonates, polystyrene-acrylonitrile copolymers and styrene-methyl methylmethacrylate copolymers, from synthetic and natural fibers, from polysaccharides and cellulose derivatives, and from glass and silicon and their derivatives.
According to preferred variants of the process of the invention,
the target nucleic acid sequence is a DNA or RNA sequence and the enzymic activities comprise RNA-dependent and/or DNA-dependent DNA polymerase activities,
the enzymic activities can additionally comprise ribonuclease H activity and DNA-dependent RNA polymerase activity in order to amplify the target nucleic acid sequence in accordance with a succession of reverse transcription, transcription and digestion reactions.
The ribonuclease H and DNA polymerase enzymic activities can be supplied by one single enzyme or else each by a different enzyme.
By way of example, the process of the invention can be employed for amplifying a target nucleic acid in a sample in accordance with techniques which are well known to the skilled person, such as PCR (polymerase chain reaction), RT-PCR (reverse transcription-polymerase chain reaction) or TMA (transcription-mediated amplification) or the NASBA (nucleic acid sequence-based amplification) technique, or any other enzymic amplification technique.
The invention also relates to a nucleotide analog or a nucleotide which is prefunctionalized and which is capable of being subjected to an enzymic treatment.
Enzymic treatment of a nucleotide analog or a nucleotide includes all the in-vivo or in-vitro reactions during which at least one enzyme, whose activity is linked to a nucleotide, is involved. Thus, it comprises all reactions which include at least one enzymic step in which a nucleotide serves as substrate for the enzyme, whether said nucleotide is transformed or not during this enzymic step; by way of example, such reactions are selected from those employed in molecular biological techniques, such as transcription, ligation, elongation and restriction and, more specifically, in amplification techniques (WINN-DEEN, Journal of Clinical Assay, Vol. 19, pp. 21-26 (1996).
Thus, the enzymes whose activities are linked to nucleotides can, in particular, be selected from the following non-exhaustive list: DNA-dependent DNA polymerases, such as the Klenow fragment of E. coli DNA polymerase I, Taq polymerase, the T7, T4 or T5 DNA polymerases, the a, b and g viral or cellular eukaryotic polymerases; RNA-dependent DNA polymerases, such as the AMV (avian myoblastosis virus) and MMLV (Moloney murine leukemia virus) polymerases; RNA polymerases, such as the T7, T3, SP6, N4 and PBSII RNA polymerases and E. coli RNA polymerase; enzymes having a nuclease activity, such as the restriction endonucleases and RNAse H; or else polyA polymerases, replicases, such as the Q-beta replicase, terminal transferases or ligases.
According to a preferred embodiment, the TMA technique for amplifying a target RNA nucleic acid sequence or amplifying a target DNA nucleic acid sequence after a reverse transcription step, with said technique being described in International Application WO 91/01384, which is hereby incorporated by reference, is chosen for applying the process of the invention.
A nucleotide analog which has been prefunctionalized in accordance with the invention corresponds to the general formula (I) 
in which
B represents a nucleobase,
Z represents a coupling arm,
n is an integer equal to 0 or 1,
X represents a covalently reactive function which is attached to at least one site in the nucleobase B,
R1 represents H or OH,
R2 represents H, OH, a monophosphate, diphosphate or triphosphate group, or an Oxe2x80x94R group in which R represents a protective group,
R3 represents H, a protective group, or a monophosphate, diphosphate or triphosphate group.
Preferably, R1 and R2 each represent, independently of the other, H or OH, and R3 represents a monophosphate, diphosphate or triphosphate group.
A prefunctionalized nucleotide of the invention is selected from the following nucleotides:
a nucleotide whose nucleobase is derived from cytosine and contains, at least on the amino function in position 4 of the pyrimidine ring, at least one nucleophilic covalently reactive function which is unprotected and which does not have a significant influence on the enzymic treatment of said nucleotide, with the covalently reactive function being selected from the NH2, Oxe2x80x94NH2, SH, hydrazine and hydrazide functions and the covalently reactive function being linked to said amino function in position 4 of the pyrimidine ring by a coupling arm which is selected from (xe2x80x94CH2xe2x80x94)n1 and (xe2x80x94Oxe2x80x94CH2xe2x80x94)n1, in which n1 is an integer of between 1 and 12; (xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94)n2, (xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94)n2, (xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94)n2 and (xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94)n2, in which n2 is an integer of between 1 and 6; and xe2x80x94NHxe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2. Advantageously, the covalently reactive function is linked to said amino function by way of a coupling arm which is selected from xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94 and NHxe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2.
a nucleotide whose base is derived from uracil and contains, at least at position 5 of the pyrimidine ring, at least one nucleophilic covalently reactive function which is unprotected and which does not significantly influence the enzymic treatment, characterized in that the covalently reactive function is selected from the NH2, Oxe2x80x94NH2, SH, hydrazine and hydrazide functions; advantageously, the covalently reactive function is linked to said amino function by way of a coupling arm which is selected from xe2x80x94Cxe2x89xa1Cxe2x80x94CH2xe2x80x94, and xe2x80x94Cxe2x89xa1Cxe2x80x94CH2xe2x80x94NHxe2x80x94COxe2x80x94CH2xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94CH2xe2x80x94NHxe2x80x94COxe2x80x94CH2xe2x80x94 and xe2x80x94CHxe2x95x90CHxe2x80x94CH2;
a nucleotide whose base is derived from adenine and contains, at least on the amino function in position 6 of the pyrimidine ring, at least one nucleophilic covalently reactive function which is unprotected and which does not have a significant influence on the enzymic treatment, characterized in that the covalently reactive function is selected from the NH2, CH2xe2x80x94Oxe2x80x94NH2, SH, hydrazine and hydrazide. functions; the covalently reactive function is linked to said amino function by way of a coupling arm which is selected from (xe2x80x94CH2xe2x80x94)n1 and (xe2x80x94Oxe2x80x94CH2xe2x80x94)n1, in which n1 represents an integer of between 1 and 12; (xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94)n2, (xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94)n2, (xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94)n2 and (xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94)n2, in which n2 is an integer of between 1 and 6; and NHxe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2. Advantageously, the covalently reactive function is linked to said amino function by way of a coupling arm which is selected from xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94, and NHxe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2; preferably, the coupling arm is xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94.
The invention also relates to the use of a prefunctionalized nucleotide, as previously defined, in an enzymic amplification treatment.
With regard to the enzymic treatment, the behavior of the prefunctionalized nucleotide analog or nucleotide is more or less identical to that of the corresponding nucleotide analog or nucleotide. This is because, as a result of its biological and chemical inertia with regard to enzymes, the function which is introduced into a site in the base of said analog or nucleotide does not significantly modify either the affinity or the specificity of the enzyme with regard to its substrate.
The invention finally relates to specific uses of nucleotides such as have just been defined. Preferably, they are used in amplification techniques such as those described in the following documents: EP-0 721 988, WO-95/03426, U.S. Pat. Nos. 5,409,818 and 5,399,491.